Thrombotic disease is a common cardiovascular and cerebrovascular disease, and includes a variety of diseases in which the stenosis and occlusion of blood vessel lumens caused by thrombus lead to ischemia and infarction of main organs, thereby causing dysfunctions, and is usually manifested by myocardial infarction, ischemic cerebral infarction and venous thromboembolism. The disease can involve various organs and systems of the whole body, and has greatly high morbidity, disability rate and mortality. About 3% of people develops different forms of thrombotic diseases each year, which severely threaten the health of human beings. According to statistics, more than 3,000,000 people die from cardiovascular and cerebrovascular diseases in our country every year, with 75% of surviving patients suffering from disability, in which more than 40% suffer from serious disability.
Currently, drugs for treating thrombotic diseases are mainly devided into three categories in clinical practice: antiplatelet drugs, thrombolytic drugs and anticlotting drugs. Among them, antiplatelet drugs refer to drugs with the function of inhibiting the adhesion, aggregation and release of platelets, which inhibit aggregation of platelets and thus avoid formation of thrombus, and can effectively prevent the development of a cardiovascular and cerebrovascular disease and prolong the survival time of a patient. Such drugs include aspirin, dipyridamole, prostacyclin, ticlopidine, a platelet membranous gpIIb/IIIa receptor antagonist, an adenosine diphosphate receptor antagonist, ozagrel, clopidogrel, clopidogrel sulfate, etc. Among them, the platelet membranous gpIIb/IIIa receptor antagonist can inhibit the aggregation of platelets through inhibiting the binding of gpIIb/IIIa receptor to fibrinogen.
Eptifibatide is one of the platelet membranous gpIIb/IIIa receptor antagonist drugs, which is a small molecular heptapeptide with a structure as shown in Formula I. It contains a modified arginine-glycine-aspartic acid (RGD) sequence, has high selectivity, can decompose quickly after blockage of the receptor, and have advantages of strong antiplatelet effect, rapid onset and less adverse reactions, thus having good market prospect.
Various impurities will be produced during the preparation of eptifibatide. The existence of impurities will have certain influence on safety and effectiveness of the drug, in which dimer impurities have greater influence on the drug quality. The dimer of eptifibatide has two structures as shown in Formulae II and III. The existence of the dimer will reduce the optical purity of the raw material of eptifibatide, and meanwhile affect the solubility thereof. Studies have shown that eptifibatide dimer impurities are mainly formed at two stages. The first stage is during the synthesis of eptifibatide. At present, a non-oriented strategy is mainly employed in synthesis of eptifibatide, in which a linear peptide is firstly prepared with solid-phase synthesis, and the linear peptide is further oxidized to obtain a cyclopeptide. During the oxidization of the linear peptide, in addition to the target product via intra-chain cyclization, dimer impurities via inter-chain cyclization will also be formed. Due to the great difference between eptifibatide and the dimer impurities thereof in properties, the dimer impurities produced during the oxidization can be removed by reverse-phase HPLC purification and other methods. The second stage generating impurities is during concentration of a refined peptide. During concentration of a refined peptide, the disulfide bond of eptifibatide easily breaks down to form an inter-dimer disulfide bond, thus forming a dimer structure. Because of the difficulty in removing the dimer impurities produced at this stage, it is especially important to determine and control eptifibatide dimer impurities produced at this stage. Up to now, there is no research report about any method of controlling the eptifibatide dimer impurities during the concentration, and it is always a challenge to figure out how dimer impurities form and how to control the formation of dimer impurities in the field of polypeptide preparation. Thus, provision of a preparation method which can control formation of eptifibatide dimers is of important and practical significance.
